JP7441002B2 - p-ethoxy nucleic acids for liposome formulations - Google Patents

Description

This application claims priority to U.S. Provisional Application No. 62/241,503, filed October 14, 2015, the entire contents of which are incorporated herein by reference.

1. FIELD OF THE INVENTION The present invention relates generally to the field of medicine. More particularly, the present invention relates to liposomal formulations of p-ethoxy oligonucleotides, and methods of making and using such formulations in medicine.

2. Description of Related Art Antisense oligonucleotides (oligos) complementary to specific regions of target mRNA have been used to inhibit endogenous gene expression. When the antisense oligonucleotide binds to the target mRNA, a DNA-RNA hybrid is formed. This hybridization inhibits translation of the mRNA and thus expression of the encoded protein. If a protein is essential for cell survival, inhibition of its expression can lead to cell death. Therefore, antisense oligonucleotides can be useful tools in anticancer and antiviral therapy.

The main obstacles to using antisense oligonucleotides to inhibit gene expression are cell instability, low cellular uptake, and low intercellular delivery. Natural phosphodiesters are not resistant to nuclease hydrolysis, which requires high concentrations of antisense oligonucleotides before any inhibitory effect is observed. Modified phosphodiester analogs such as p-ethoxy have been created to overcome this nuclease hydrolysis problem, but these have not provided a satisfactory solution to the problem.

Cellular uptake of antisense oligonucleotides is low. To solve this problem, physical techniques such as calcium phosphate precipitation, DEAE-dextran-mediated, or electroporation have been used to increase cellular uptake of oligonucleotides. These techniques are difficult to reproduce and are not applicable in vivo. Cationic lipids such as lipofectin have also been used to deliver oligonucleotides. Electrostatic interactions are formed between the cationic lipid and the negatively charged oligonucleotide, resulting in a complex that is subsequently taken up by the target cell. These cationic lipids are harmful to cell membranes because they do not protect oligonucleotides from nuclease digestion, and are only useful in the delivery of nuclease-resistant phosphorothioates, but not in the delivery of nuclease-cleavable phosphodiesters. .

Another modified phosphodiester (PD) analog that has been prepared is p-ethoxy (pE) oligo. Modifications of pE oligos are made on the phosphate backbone such that the modifications do not interfere with the binding of these oligos to the target mRNA. pE oligos are made by adding ethyl groups to the non-bridging oxygen atoms of the phosphate backbone, thereby making these oligos uncharged compounds. Despite their resistance to nucleases, upon internalization these oligos remain sequestered inside the vacuole of endosomes/lysosomes, and in order to block their access to the target mRNA, pE oligos Cellular uptake and intracellular delivery of is poor.

There is a need for improved antisense compositions for use in treating disease, as well as improved processes for making such compositions.

In one embodiment, a composition comprising a population of oligonucleotides is provided. In some embodiments, the oligonucleotides of the population are comprised of nucleoside molecules linked to each other through phosphate backbone bonds, and at least one of the phosphate backbone bonds in each oligonucleotide is a p-ethoxy backbone bond. and 80% or less of the phosphate backbone bonds in each oligonucleotide are p-ethoxy backbone bonds. In some embodiments, at least one of the phosphate backbone linkages in each oligonucleotide is a phosphodiester backbone linkage. In some embodiments, 10% to 80% of the phosphate backbone bonds are p-ethoxy backbone bonds, and 20% to 80% of the phosphate backbone bonds are p-ethoxy backbone bonds, and the phosphate backbone bonds are p-ethoxy backbone bonds. Of these, 30% to 80% are p-ethoxy backbone bonds, 40% to 80% of phosphate backbone bonds are p-ethoxy backbone bonds, and 50% to 80% of phosphate backbone bonds. are p-ethoxy backbone bonds, or 60% to 70% of the phosphate backbone bonds are p-ethoxy backbone bonds, or any range derivable therein. In some embodiments, 20% to 90% of the phosphate backbone linkages are phosphodiester backbone linkages, 20% to 80% of the phosphate backbone linkages are phosphodiester backbone linkages, and 20% to 90% of the phosphate backbone linkages are phosphodiester backbone linkages; Of these, 20% to 70% are phosphodiester backbone bonds, 20% to 60% of phosphate backbone bonds are phosphodiester backbone bonds, and 20% to 50% of phosphate backbone bonds are phosphodiester backbone bonds. They are diester backbone bonds, or 30% to 40% of the phosphate backbone bonds are phosphodiester backbone bonds, or any range that can be derived therein. In various embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65 of the phosphate backbone linkages %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any value therein, is a p-ethoxy backbone bond. In various embodiments, up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% of the phosphate backbone linkages, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any value therein, are phosphodiester backbone linkages. In some embodiments, the composition is lyophilized.

In some embodiments, the population of oligonucleotides has a size ranging from 7 to 30 nucleotides. In certain embodiments, the oligonucleotides of the population have a size ranging from 12 to 25 unique nucleotides. In various aspects, the population of oligonucleotides is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, It has a size of 24, 25, 26, 27, 28, 29, or 30 nucleotides. This size range may be the average size of oligonucleotides in the population.

In some embodiments, the oligonucleotides of the population are 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, have an average size of 26, 27, 28, 29, or 30 nucleotides, with 5, 6, 7, 8, 8, 9, 10, 11 of the phosphate backbone linkages in each oligonucleotide , 12, 13, 14, 15, 15, 16, 17, 18, 19, 20, 20, 21, 22, 23, or 24 or less are p-ethoxy skeleton bonds, respectively. In some embodiments, the oligonucleotides of the population are 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, having an average size of 26, 27, 28, 29, or 30 nucleotides, and at least 2, 2, 2, 2, 3, 3, respectively, of the phosphate backbone linkages in each oligonucleotide; 3, 3, 4, 4, 4, 4, 4, 5, 5, 5, 5, 5, 5, 6, 6, 6, 6, or 6 is a phosphodioester backbone bond.

In some embodiments, the population of oligonucleotides comprises a single species of oligonucleotide. In other embodiments, the population of oligonucleotides includes at least two oligonucleotides. Oligonucleotides of a single species can have the same nucleotide sequence, but either have p-ethoxy linkages at different positions within the molecule or lack them. In some embodiments, the population of oligonucleotides includes antisense oligonucleotides, short interfering RNAs (siRNAs), microRNAs (miRNAs), or piwiRNAs (piRNAs).

In certain embodiments, the population of oligonucleotides inhibits expression of at least one oncogenic protein, infectious agent protein, or autoantigen. In some embodiments, the oligonucleotides of the population hybridize with at least one oncogenic oligonucleotide, infectious agent oligonucleotide, or autoantigen oligonucleotide.

In various embodiments, the composition further comprises a phospholipid. In some embodiments, the phospholipid is uncharged or has a neutral charge at physiological pH. In some embodiments, the phospholipid is a neutral phospholipid. In certain embodiments, the neutral phospholipid is phosphatidylcholine. In certain embodiments, the neutral phospholipid is dioleoylphosphatidylcholine. In some embodiments, the phospholipids are essentially cholesterol-free.

In some embodiments, the phospholipid and oligonucleotide are present in a molar ratio of about 5:1 to about 100:1, or any ratio derivable therein. In various embodiments, the phospholipid and oligonucleotide are about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50 :1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1 molar ratio. In some embodiments, the oligonucleotide and phospholipid form an oligonucleotide-lipid complex, such as a liposome complex. In some embodiments, at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% of the liposomes, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% are less than 5 microns in diameter. In various embodiments, the composition further comprises at least one surfactant, such as, for example, polysorbate 20. In some embodiments, at least about 5% of the total liposomal p-ethoxy antisense formulation consists of surfactant and at least about 90% of the liposomes are less than 5 microns in diameter. In some embodiments, at least about 15% of the total liposomal p-ethoxy antisense medical product is comprised of surfactant, and at least about 90% of the liposomes are less than 3 microns in diameter. In some embodiments, the population of oligonucleotides is incorporated into a population of liposomes.

In one aspect, the population of oligonucleotides each comprises a length of about 21 nucleotides and has about 30% phosphodiester backbone linkages. In one aspect, the population of oligonucleotides may be further incorporated into a liposome composition comprising at least about 5% surfactant, wherein at least about 90% of the liposomes have a diameter of less than about 5 microns. It is possible.

In one embodiment, a pharmaceutical composition is provided that includes an oligonucleotide and phospholipid composition of this embodiment and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a chemotherapeutic agent.

In one embodiment, a method is provided for delivering a therapeutically effective amount of an oligonucleotide to a cell, comprising contacting the cell with a pharmaceutical composition of this embodiment. In some embodiments, the method is a method of treating hyperplasia, cancer, autoimmune disease, or infectious disease.

In one embodiment, a method is provided for treating a subject with cancer, an autoimmune disease, or an infectious disease, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of this embodiment. In some embodiments, the subject is a human. In some embodiments, the cancer is in the bladder, blood, pancreas, bone, bone marrow, brain, breast, colon, esophagus, stomach, head and neck, kidneys, liver, lungs, prostate, skin, testes, tongue, ovaries. , or uterine cancer. In some embodiments, the autoimmune disease is lupus erythematosus, Sjögren's disease, Crohn's disease, diabetes mellitus, multiple sclerosis, or rheumatoid arthritis. In some embodiments, the infectious disease is a bacterial, fungal, viral, or parasitic infection. In some embodiments, the composition is administered subcutaneously, intravenously, or intraperitoneally. In some embodiments, the method further comprises administering to the subject at least a second anti-cancer therapy. In some embodiments, the second anti-cancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, or cytokine therapy.

Oligonucleotides include antisense nucleic acid molecules that specifically hybridize to nucleic acid molecules that encode or modulate the expression of a target protein. "Specific hybridization" means that an antisense nucleic acid molecule hybridizes to a targeted nucleic acid molecule and modulates its expression. Preferably, "specific hybridization" also means that other genes or transcripts are not affected. The oligonucleotide can be a single-stranded nucleic acid, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, It may contain 25, 26, 27, 28, 29, 30 or more nucleobases. In certain aspects, the oligonucleotide can include 15-30, 19-25, 20-23, or 21 contiguous nucleobases. In certain embodiments, the oligonucleotide inhibits translation of a gene that promotes the proliferation of cancerous or pre-cancerous or hyperplastic mammalian cells (eg, human cells). Oligonucleotides can induce apoptosis and/or inhibit translation of oncogenes or other target genes within cells. In certain embodiments, the oligonucleotide component comprises a single species of oligonucleotide. In other embodiments, the oligonucleotide component includes 2, 3, 4, or more types of oligonucleotides that target 1, 2, 3, 4, or more genes. The compositions may further include chemotherapeutic or other anti-cancer agents, which may or may not be incorporated into the lipid components or liposomes of the invention. In further embodiments, the oligonucleotide component is incorporated within a liposome or lipid component.

"Capture," "encapsulate," and "incorporate" refer to the liposome forming a blockage of free diffusion into solution by association with or around a drug of interest, e.g. Liposomes can encapsulate the drug within the lipid layer or within an aqueous compartment within or between the lipid layers. In certain embodiments, the composition is contained in a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be formulated for administration to human subjects or patients.

In certain embodiments, the lipid component has an essentially neutral charge, such as comprising a neutral phospholipid or a net neutral charge. In certain embodiments, the neutral phospholipid is DOPC, egg phosphatidylcholine ("EPC"), dilauriloyl phosphatidylcholine ("DLPC"), dimyristoyl phosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), Distearoylphosphatidylcholine (“DSPC”), 1-myristoyl-2-palmitoylphosphatidylcholine (“MPPC”), 1-palmitoyl-2-myristoylphosphatidylcholine (“PMPC”), 1-palmitoyl-2-stearoylphosphatidylcholine (“PSPC”) , 1-stearoyl-2-palmitoylphosphatidylcholine (“SPPC”), dimyristylphosphatidylcholine (“DMPC”), 1,2-distearoyl-sn-glycero-3-phosphocholine (“DAPC”), 1,2-diarachidyl- sn-glycero-3-phosphocholine (“DBPC”), 1,2-diacosenoyl-sn-glycero-3-phosphocholine (“DEPC”), palmitoyloleoylphosphatidylcholine (“POPC”), lysophosphatidylcholine, or dilinoleoylphosphatidylcholine It can be a phosphatidylcholine such as. In other aspects, the neutral phospholipid is dioleoylphosphatidylethanolamine (“DOPE”), distearoylphosphatidylethanolamine (“DSPE”), dimyristoylphosphatidylethanolamine (“DMPE”), dipalmitoylphosphatidylethanolamine ("DPPE"), palmitoyloleoylphosphatidylethanolamine ("POPE"), or lysophosphatidylethanolamine. In certain embodiments, the phospholipid component may include 1, 2, 3, 4, 5, 6, 7, 8 or more types or types of neutral phospholipids. In other embodiments, the phospholipid component may include 2, 3, 4, 5, 6 or more types or types of neutral phospholipids.

In certain embodiments, the lipid component may have an essentially neutral charge, since it includes positively charged lipids and negatively charged lipids. The lipid component may further include neutrally charged lipid(s) or phospholipid(s). The positively charged lipid may be a positively charged phospholipid. The negatively charged lipid may be a negatively charged phospholipid. The negatively charged phospholipid can be a phosphatidylserine, such as dimyristoylphosphatidylserine (“DMPS”), dipalmitoylphosphatidylserine (“DPPS”), or brain phosphatidylserine (“BPS”). Negatively charged phospholipids include dilauriloyl phosphatidylglycerol (“DLPG”), dimyristoyl phosphatidylglycerol (“DMPG”), dipalmitoylphosphatidylglycerol (“DPPG”), distearoyl phosphatidylglycerol (“DSPG”), or diole It can be a phosphatidylglycerol, such as oleophosphatidylglycerol ("DOPG"). In certain embodiments, the composition further comprises cholesterol or polyethylene glycol (PEG). In other embodiments, the composition is essentially cholesterol-free. In certain embodiments, the phospholipid is a naturally occurring phospholipid. In other embodiments, the phospholipid is a synthetic phospholipid.

Liposomes can be made from one or more phospholipids so long as the lipid material is substantially uncharged. It is important that the composition is substantially free of anionic and cationic phospholipids and cholesterol. Suitable phospholipids include phosphatidylcholine and others well known to those skilled in the art.

Another aspect of the invention involves a method for delivering an oligonucleotide to a cell comprising contacting the cell with a neutral lipid composition of the invention. The method will provide an effective amount of the composition of the invention. An effective amount is an amount of a therapeutic component that attenuates, slows, reduces or eliminates a cell, pathology or disease state in a subject. The cells can be comprised in a subject, such as a human, or a patient. The methods can further include methods of treating cancer or other hyperplastic conditions. Cancer may be in the bladder, blood, bones, bone marrow, brain, chest, colon, esophagus, gastrointestinal tract, gums, head, kidneys, liver, lungs, nasopharynx, neck, prostate, skin, stomach, testes, tongue, or May originate from the uterus. In certain embodiments, the method further includes a method of treating a non-cancerous disease or hyperplastic condition. Cells may be pre-cancerous or cancerous. In certain embodiments, the compositions and methods inhibit cell proliferation, induce apoptosis in cells, and/or inhibit oncogene translation. Oligonucleotides can inhibit translation of genes that are overexpressed in cancerous cells.

In certain embodiments, the methods of the invention further include administering additional therapy to the subject. Additional therapy may include administering chemotherapeutic agents (eg, paclitaxel or docetaxel), surgery, radiation therapy, and/or gene therapy. In certain embodiments, the chemotherapy is docetaxel, paclitaxel, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, Doxorubicin, bleomycin, pricomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binders, taxol, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine, methotrexate, or It is a combination of these. In certain embodiments, the chemotherapy is a taxane, such as docetaxel or paclitaxel. Chemotherapy can be delivered to the neutral lipid compositions of the invention before, during, after, or a combination thereof. Chemotherapy may be delivered within 0, 1, 5, 10, 12, 20, 24, 30, 48, or 72 hours or longer of delivery of the neutral lipid composition. The neutral lipid composition, the second anti-cancer therapy, or both the neutral lipid composition and the anti-cancer therapy can be administered intratumorally, intravenously, intraperitoneally, subcutaneously, orally, or various combinations thereof.

It is envisioned that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, the compositions of the invention can be used to accomplish the methods of the invention.

As used herein, "essentially free" with respect to a particular ingredient means that none of the particular ingredient is intentionally incorporated into the composition and/or as a contaminant; Used to mean present only in trace amounts. Thus, the total amount of specific components due to unintentional contamination of the composition is much less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of a particular component cannot be detected by standard analytical methods.

As used herein, "a" or "an" may mean one or more. As used herein in the claim(s), the word "a" or "an" when used in conjunction with the word "comprising" means one or more. There is.

The use of the term "or" in the claims is used to mean "and/or" unless it is expressly indicated to refer only to alternatives or the alternatives are mutually exclusive. However, this disclosure supports alternatives and definitions that refer only to "and/or." As used herein, "another" may mean at least a second or more.

Throughout this application, the term "about" is used to indicate that a value includes the inherent variation in error for the equipment, and the method is intended to include this value, or the variation that exists between test subjects. used to make decisions.

Other objects, features and advantages of the invention will become apparent from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, as various changes within the spirit and scope of the invention may be made. Changes and modifications will become apparent to those skilled in the art from this detailed description.
The present invention provides, for example, the following items.
(Item 1)
A composition comprising a population of oligonucleotides, wherein the oligonucleotides in the population are composed of nucleoside molecules linked together through phosphate backbone bonds, and wherein at least one of the phosphate backbone bonds in each oligonucleotide is One of the aforementioned compositions is a p-ethoxy backbone bond, and 80% or less of the phosphate backbone bonds in each oligonucleotide are p-ethoxy backbone bonds.
(Item 2)
The composition according to item 1, wherein 10% to 80% of the phosphate backbone bonds are p-ethoxy backbone bonds.
(Item 3)
The composition according to item 2, wherein 20% to 80% of the phosphate backbone bonds are p-ethoxy backbone bonds.
(Item 4)
The composition according to item 3, wherein 30% to 80% of the phosphate backbone bonds are p-ethoxy backbone bonds.
(Item 5)
The composition according to item 4, wherein 40% to 80% of the phosphate backbone bonds are p-ethoxy backbone bonds.
(Item 6)
The composition according to item 5, wherein 50% to 80% of the phosphate backbone bonds are p-ethoxy backbone bonds.
(Item 7)
The composition according to item 6, wherein 60% to 70% of the phosphate backbone bonds are p-ethoxy backbone bonds.
(Item 8)
The composition according to item 1, wherein 20% to 90% of the phosphate backbone bonds are phosphodiester backbone bonds.
(Item 9)
The composition according to item 8, wherein 20% to 80% of the phosphoric acid backbone bonds are phosphodiester backbone bonds.
(Item 10)
The composition according to item 9, wherein 20% to 70% of the phosphoric acid backbone bonds are phosphodiester backbone bonds.
(Item 11)
The composition according to item 10, wherein 20% to 60% of the phosphoric acid backbone bonds are phosphodiester backbone bonds.
(Item 12)
The composition according to item 11, wherein 20% to 50% of the phosphoric acid backbone bonds are phosphodiester backbone bonds.
(Item 13)
13. The composition according to item 12, wherein 30% to 40% of the phosphoric acid backbone bonds are phosphodiester backbone bonds.
(Item 14)
The composition of item 1, wherein the oligonucleotides of the population have a size ranging from 7 to 30 nucleotides.
(Item 15)
15. The oligonucleotides of the population have an average size of 7 nucleotides, and no more than 5 of the phosphate backbone linkages in each oligonucleotide are p-ethoxy backbone linkages. Composition.
(Item 16)
15. The oligonucleotides of the population have an average size of 10 nucleotides, and no more than 8 of the phosphate backbone linkages in each oligonucleotide are p-ethoxy backbone linkages. Composition.
(Item 17)
15. The oligonucleotides of the population have an average size of 30 nucleotides, and no more than 24 of the phosphate backbone linkages in each oligonucleotide are p-ethoxy backbone linkages. Composition.
(Item 18)
15. The composition of item 14, wherein the oligonucleotides of the population have a size ranging from 12 to 25 nucleotides.
(Item 19)
19. The oligonucleotides of the population have an average size of 15 nucleotides, and no more than 12 of the phosphate backbone linkages in each oligonucleotide are p-ethoxy backbone linkages. Composition.
(Item 20)
19. The oligonucleotides of the population have an average size of 18 nucleotides, and no more than 14 of the phosphate backbone linkages in each oligonucleotide are p-ethoxy backbone linkages. Composition.
(Item 21)
19. The oligonucleotides of the population have an average size of 20 nucleotides, and no more than 16 of the phosphate backbone linkages in each oligonucleotide are p-ethoxy backbone linkages. Composition.
(Item 22)
19. The oligonucleotides of the population have an average size of 25 nucleotides, and no more than 20 of the phosphate backbone linkages in each oligonucleotide are p-ethoxy backbone linkages. Composition.
(Item 23)
2. The composition of item 1, wherein the population of oligonucleotides comprises a single species of oligonucleotide.
(Item 24)
The composition of item 1, wherein the population of oligonucleotides comprises at least two species of oligonucleotides.
(Item 25)
The composition of item 1, wherein the population of oligonucleotides comprises antisense oligonucleotides, short interfering RNAs, microRNAs, or piwiRNAs.
(Item 26)
2. The composition of item 1, wherein said oligonucleotides of said population inhibit the expression of at least one oncogenic protein, infectious agent protein, or autoantigen.
(Item 27)
The composition of item 1, wherein the oligonucleotides of the population hybridize with at least one oncogenic oligonucleotide, infectious agent oligonucleotide, or autoantigen oligonucleotide.
(Item 28)
The composition according to item 1, further comprising a phospholipid, wherein the oligonucleotide and the phospholipid form an oligonucleotide-lipid complex.
(Item 29)
29. The composition of item 28, wherein the phospholipid is uncharged or has a neutral charge at physiological pH.
(Item 30)
The composition according to item 29, wherein the phospholipid is a neutral phospholipid.
(Item 31)
The composition according to item 30, wherein the neutral phospholipid is phosphatidylcholine.
(Item 32)
31. The composition according to item 30, wherein the neutral phospholipid is dioleoylphosphatidylcholine.
(Item 33)
29. The composition of item 28, wherein the phospholipid is essentially free of cholesterol.
(Item 34)
29. The composition of item 28, wherein the phospholipid and oligonucleotide are present in a molar ratio of about 5:1 to about 100:1.
(Item 35)
29. The composition of item 28, wherein the oligonucleotide-lipid complex is further defined as a population of liposomes.
(Item 36)
36. The composition of item 35, wherein at least 90% of the liposomes are less than 5 microns in diameter.
(Item 37)
36. The composition of item 35, wherein said population of oligonucleotides is incorporated into said population of liposomes.
(Item 38)
The composition of item 1, wherein the composition is lyophilized.
(Item 39)
A pharmaceutical composition comprising the composition according to item 28 and a pharmaceutically acceptable carrier.
(Item 40)
40. The composition of item 39, further comprising a chemotherapeutic agent.
(Item 41)
40. A method for delivering a therapeutically effective amount of an oligonucleotide to a cell, said method comprising contacting said cell with a pharmaceutical composition according to item 39.
(Item 42)
42. The method of item 41, wherein the method is a method of treating hyperplasia, cancer, autoimmune disease, or infectious disease.
(Item 43)
40. A method of treating a subject having cancer, an autoimmune disease, or an infectious disease, said method comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition according to item 39.
(Item 44)
44. The method according to item 43, wherein the subject is a human.
(Item 45)
An item in which the cancer is bladder, blood, pancreas, bone, bone marrow, brain, breast, colon, esophagus, stomach, head and neck, kidney, liver, lung, prostate, skin, testicular, tongue, ovarian, or uterine cancer. 43.
(Item 46)
44. The method of item 43, wherein the autoimmune disease is lupus erythematosus, Sjögren's disease, Crohn's disease, diabetes mellitus, multiple sclerosis, or rheumatoid arthritis.
(Item 47)
44. The method according to item 43, wherein the infectious disease is a bacterial infection, a fungal infection, a viral infection, or a parasitic infection.
(Item 48)
44. The method of item 43, wherein the composition is administered subcutaneously, intravenously, or intraperitoneally.
(Item 49)
44. The method of item 43, further comprising administering at least a second anti-cancer therapy to the subject.
(Item 50)
The second anticancer therapy may include surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, antiviral therapy, immunosuppressive therapy, antibacterial therapy, antiparasitic therapy, antifungal therapy, or cytokine therapy. The method of item 49, which is therapy.

Description of Exemplary Embodiments The present invention provides compositions and methods for the delivery of oligonucleotides (e.g., inhibitors of gene expression) to cells via lipid compositions, and in certain aspects, about A lipid composition having zero net charge, ie, a neutral lipid composition is provided. In certain embodiments, the lipid composition is an uncharged liposome. These methods can be used effectively to treat cancer.

I. Lipids and Liposomes "Liposome" is used herein to mean lipid-containing vesicles having a lipid bilayer, as well as other lipid carrier particles that can entrap or incorporate antisense oligonucleotides. Thus, liposome is a general term encompassing a variety of unilamellar, multilamellar, and multivesicular lipid vehicles formed by the production of encapsulated lipid bilayers or aggregates. Additionally, liposomes may have an undefined lamellar structure. Liposomes may be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in excess aqueous solution, multilamellar liposomes form spontaneously. The lipid components undergo self-rearrangement before forming a closed structure and trap water and solutes dissolved between the lipid bilayers (Ghosh and Bachhawat, 1991). However, the invention also encompasses compositions that have structures in solution that differ from normal vesicular structures. For example, lipids may adopt a micellar structure or simply exist as aggregates of heterogeneous lipid molecules.

Liposomes are a form of nanoparticles that are carriers for delivering various drugs into diseased tissues. Optimal liposome size depends on the target tissue. In tumor tissue, the vasculature is discontinuous and the pore size varies from 100 to 780 nm (Siwak et al., 2002). In comparison, pore size in normal vascular endothelium is less than 2 nm in most tissues and 6 nm in postcapillary venules. Negatively charged liposomes are thought to be cleared from the circulation more rapidly than neutral or positively charged liposomes; however, recent studies have shown that negatively charged lipid types are It has been shown to affect the rate of liposome uptake. For example, liposomes containing negatively charged lipids (phosphatidylserine, phosphatidic acid, and phosphatidylglycerol) that are not sterically shielded are cleared more quickly than neutral liposomes. Interestingly, cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]) and cationic liposome-DNA complexes were anionic, neutral, or sterically stabilized. They are more strongly bound and internalized by endothelial cells of angiogenic blood vessels via endocytosis than neutral liposomes (Thurston et al., 1998; Krasnici et al., 2003). Cationic liposomes are ideal delivery vehicles for tumor cells because their surface interactions with tumor cells create an electrostatically induced binding site barrier effect that inhibits further association of the delivery system with tumor spheroids. (Kostarelos et al., 2004). However, neutral liposomes appear to have better intratumoral penetration. Toxicity with certain liposome preparations is also a concern. Cationic liposomes induce dose-dependent toxicity and pulmonary inflammation by promoting the release of reactive oxygen intermediates, and this effect is more pronounced in polycationic liposomes than in monovalent cationic liposomes such as DOTAP. (Dokka et al., 2000). Neutral and negatively charged liposomes do not appear to present pulmonary toxicity (Guitierrez-Puente et al., 1999). Although cationic liposomes efficiently uptake nucleic acids, they have had limited success in gene downregulation in vivo, likely due to their stable intracellular nature and the resulting failure to release the nucleic acid contents. Neutral-charged lipids, or neutralized-charged lipid compositions, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), have neutral properties and in vivo antisense oligo As used herein for successful delivery of nucleotides.

The present invention provides methods and compositions for associating oligonucleotides, such as antisense oligonucleotides, with lipids and/or liposomes. The oligonucleotide is incorporated into the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, linked to the liposome via binding molecules that are associated with both the liposome and the oligonucleotide, and is entrapped within the liposome. dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, containing micelles, or forming complexes with or can be associated with lipids in other ways. The liposomes or liposome/oligonucleotide associated compositions provided herein are not limited to any particular structure in solution. For example, they may exist in bilayer structures, as micelles, or with "collapsible" structures. They may simply be scattered in the solution and may form aggregates that are not uniform in size or shape.

A. Lipids Lipids are fatty substances that may be of natural origin or synthetic. For example, lipids include naturally occurring lipid droplets in the cytoplasm, as well as long chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes, which are well known to those skilled in the art. Includes classes of compounds. An example is the lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

Lipid compositions of the invention may include phospholipids. In certain embodiments, a single species or type of phospholipid may be used to create lipid compositions such as liposomes. In other embodiments, one or more than one type of phospholipid may be used.

Phospholipids include glycerophospholipids and certain sphingolipids. Phospholipids include, but are not limited to: dioleoylphosphatidylcholine (“DOPC”), egg phosphatidylcholine (“EPC”), dilauriloylphosphatidylcholine (“DLPC”), dimyristoyl phosphatidylcholine (“ DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), 1-palmitoyl-2-myristoylphosphatidylcholine ("PMPC"), 1-palmitoyl-2-stearoylphosphatidylcholine (“PSPC”), 1-stearoyl-2-palmitoylphosphatidylcholine (“SPPC”), dilauriloylphosphatidylglycerol (“DLPG”), dimyristoylphosphatidylglycerol (“DMPG”), Palmitoylphosphatidylglycerol (“DPPG”), Distearoylphosphatidylglycerol (“DSPG”), Distearoylsphingomyelin (“DSSP”), Distearoylphosphatidylethanolamine (“DSPE”), Dioleoylphosphatidylglycerol (“DOPG”) , dimyristoyl phosphatidic acid (“DMPA”), dipalmitoylphosphatidic acid (“DPPA”), dimyristoyl phosphatidylethanolamine (“DMPE”), dipyristoyl phosphatidylethanolamine (“DPPE”), dimyristoyl phosphatidylserine (“DMPS”) ”), dipalmitoylphosphatidylserine (“DPPS”), brain phosphatidylserine (“BPS”), brain sphingomyelin (“BSP”), dipalmitoylphosphingomyelin (“DPSP”), dimyristylphosphatidylcholine (“DMPC”) ), 1,2-distearoyl-sn-glycero-3-phosphocholine (“DAPC”), 1,2-dialachidoyl-sn-glycero-3-phosphocholine (“DBPC”), 1,2-diacosenoyl-sn-glycero -3-phosphocholine (“DEPC”), dioleoylphosphatidylethanolamine (“DOPE”), palmitoyloleoylphosphatidylethanolamine (“POPC”), palmitoyloleoylphosphatidylethanolamine (“POPE”), lysophosphatidylcholine, lysophosphatidylethanol amines, and dilinoleoylphosphatidylcholines.

Phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine, because phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., approximately pH 7), so these compounds can be used in neutral liposomes. may be particularly useful for producing . In certain embodiments, the phospholipid DOPC is used to generate uncharged liposomes or lipid compositions. In certain embodiments, lipids that are not phospholipids (e.g., cholesterol) can also be used.

Phospholipids may be derived from natural or synthetic sources. However, in certain embodiments, phospholipids derived from natural sources, such as egg or soy phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol, heart cardiolipin, and plant or bacterial phosphatidylethanolamine, can be added to the resulting liposomes. It is not used as a primary phosphatide (ie, making up 50% or more of the total phosphatide composition) because it can lead to instability and leakage.

B. Neutral Liposome "Neutral liposome or lipid composition" or "uncharged liposome or lipid composition" as used herein produces an essentially neutral net charge (substantially uncharged) Defined as a liposome or lipid composition with one or more lipids. In certain embodiments, neutral liposomes or lipid compositions can include lipids and/or phospholipids that are largely neutral themselves. In certain embodiments, amphiphilic lipids can be incorporated into or used to produce neutral liposomes or lipid compositions. For example, neutral liposomes can be produced by combining positively and negatively charged lipids such that these charges substantially cancel each other out, thereby producing an essentially neutral net charge. Do it like this. "Essentially neutral" or "essentially uncharged" means that the few, if any, lipids within a given population (e.g., a population of liposomes) are affected by the opposite charge of another component. Contains uncanceled charges (eg, less than 10% of the components, more preferably less than 5%, most preferably less than 1% of the components contain uncanceled charges). In certain embodiments of the invention, compositions may be prepared in which the lipid component of the composition is essentially neutral, but not in the form of liposomes.

The size of liposomes varies depending on the method of synthesis. Liposomes suspended in aqueous solution are generally in the shape of spherical vesicles and may have one or more concentric layers of lipid bilayer molecules. Each layer consists of a parallel array of molecules of the formula XY, where X is a hydrophilic moiety and Y is a hydrophobic moiety. In aqueous suspensions, the concentric layers are arranged such that the hydrophilic portions remain in contact with the aqueous phase and the hydrophobic regions tend to self-associate. For example, when an aqueous phase is present both inside and outside the liposome, lipid molecules can form bilayers known as lamellae with an arrangement of XY-YX. Lipid aggregates can be formed when the hydrophilic and hydrophobic parts of two or more lipid molecules become associated with each other. The size and shape of these aggregates will depend on many different variables, such as the nature of the solvent and the presence of other compounds in the solution.

Liposomes within the scope of the invention are described, for example, in Bangham et al., the contents of which are incorporated herein by reference. (1965), the method of Gregoriadis (1979), the contents of which are incorporated herein by reference, the method of Deamer and Uster (1983), the contents of which are incorporated herein by reference, and the method of Szoka and Papahadjopoulos (1978), the contents of which are incorporated herein by reference. ) can be prepared according to known experimental techniques, such as the reversed-phase evaporation method as described by (1999). The methods described above differ in their respective ability to capture aqueous material and their respective aqueous space to lipid ratio.

In certain embodiments, neutral liposomes may be used to deliver oligonucleotides, such as antisense oligonucleotides. Neutral liposomes may contain a single species of oligonucleotide directed to inhibit the translation of a single gene, or neutral liposomes may contain multiple oligonucleotides directed to inhibit the translation of multiple genes. may contain oligonucleotides of the following species. Additionally, neutral liposomes can contain a chemotherapeutic agent in addition to the oligonucleotide, whereby, in certain embodiments, the chemotherapeutic agent and the oligonucleotide are in the same composition or in separate compositions. , to cells (eg, cancerous cells of a human subject).

Dry lipids or lyophilized liposomes can be dehydrated and reconstituted at the appropriate concentration using a suitable solvent (eg, DPBS or Hepes buffer). The mixture can then be shaken vigorously in a vortex mixer. Liposomes can be resuspended at an appropriate total phospholipid concentration (eg, about 10-200 mM). Unencapsulated oligonucleotides can be removed by centrifugation at 29,000 g and the liposome pellet washed. Alternatively, unencapsulated oligonucleotide can be removed by dialysis against excess solvent. The amount of encapsulated oligonucleotide can be determined according to standard methods.

II. Inhibition of Gene Expression Inhibitory oligonucleotides can inhibit the transcription or translation of genes in cells. Oligonucleotides may be 5-50 or more nucleotides in length, and in certain embodiments 7-30 nucleotides in length. In certain embodiments, the oligonucleotide is 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26 , 27, 28, 29 or 30 nucleotides in length. Oligonucleotides may include nucleic acids and/or nucleic acid analogs. Typically, an inhibitory oligonucleotide will inhibit the translation of a single gene within a cell, but in certain embodiments, an inhibitory oligonucleotide will inhibit the translation of two or more genes within a cell. It may also impede translation.

Within an oligonucleotide, the components of the oligonucleotide need not be of the same type or uniform throughout (eg, an oligonucleotide may include nucleotides and nucleic acids, or nucleotide analogs). In certain embodiments of the invention, oligonucleotides may include only a single nucleic acid, or nucleic acid analog. 5,6,7,8,9,10,11,12,13,14,15,16,17,18, the inhibitory oligonucleotide hybridizes with a complementary nucleic acid to form a double-stranded structure It may contain 19, 20, 25, 30, or more contiguous nucleobases, including all ranges therebetween.

III. Nucleic Acids The present invention provides methods and compositions for the delivery of oligonucleotides via neutral liposomes. Because oligonucleotides are composed of nucleic acids, methods involving nucleic acids (eg, production of nucleic acids, modification of nucleic acids, etc.) can also be used with respect to oligonucleotides.

The term "nucleic acid" is well known in the art. "Nucleic acid" as used herein generally refers to a molecule (ie, a chain) of DNA, RNA, or a derivative or analog thereof, that includes a nucleobase. These definitions refer to single-stranded or double-stranded nucleic acids. Although double-stranded nucleic acids may be formed by completely complementary binding, in some embodiments double-stranded nucleic acids may be formed by partially or substantially complementary binding. As used herein, single-stranded nucleic acids may be designated by the prefix "ss" and double-stranded nucleic acids may be designated by the prefix "ds."

A. Nucleobases As used herein, "nucleobases" refer to naturally occurring nucleobases (i.e., A, T, G, C or U), and derivative(s) of natural or non-natural origin, and analogs of such nucleobases. Nucleobases generally include at least one naturally occurring nucleobase in a manner that can replace the formation of naturally occurring nucleobase pairs (e.g., hydrogen bonds between A and T, G and C, and A and U). can form one or more hydrogen bonds with (i.e., "anneal" or "hybridize"). Nucleobases can be included in nucleosides or nucleotides using any chemical or natural synthetic method described herein or known to those skilled in the art.

"Purine" and/or "pyrimidine" nucleobase(s) include one or more alkyl, carboxyalkyl, amino, hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol, or alkylthiol moieties. includes naturally occurring purine and/or pyrimidine nucleobases, and derivative(s) and analog(s) thereof, including, but not limited to, purines or pyrimidines substituted with . Preferred alkyl (eg, alkyl, carboxyalkyl, etc.) moieties contain from about 1, about 2, about 3, about 4, about 5 to about 6 carbon atoms. Other non-limiting examples of purines or pyrimidines include deazapurine, 2,6-diaminopurine, 5-fluorouracil, xanthine, hypoxanthine, 8-bromoguanine, 8-chloroguanine, bromothyline, 8-aminoguanine, 8 -Hydroxyguanine, 8-methylguanine, 8-thioguanine, azaguanine, 2-aminopurine, 5-ethylcytosine, 5-methylcytosine, 5-bromouracil, 5-ethyluracil, 5-iodouracil, 5-chlorouracil, 5-propyluracil, thiouracil, 2-methyladenine, methylthioadenine, N,N-dimethyladenine, azaadenine, 8-bromoadenine, 8-hydroxyadenine, 6-hydroxyaminopurine, 6-thiopurine, 4-(6-amino hexyl/cytosine), etc. Purine and pyrimidine derivatives or analogs include (with omitted/modified base description) ac4c/4-acetylcytidine, Mam5s2u/5-methoxyaminomethyl-2-thiouridine, Chm5u/5-(carboxyhydroxylmethyl)uridine , Manq/beta, D-mannosyl eosin, Cm/2'-O-methylcytidine, Mcm5s2u/5-methoxycarbonylmethyl-2-thiouridine, Cmnm5s2u/5-carboxymethylamino-methyl-2-thioridine, Mcm5u/5 -Methoxycarbonylmethyluridine, Cmnm5u/5-carboxymethylaminomethyluridine, Mo5u/5-methoxyuridine, D/dihydrouridine, Ms2i6a, 2-methylthio-N6-isopentenyladenosine, Fm/2'-O-methylpseudouridine , Ms2t6a/N-((9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine, Gal q/β, D-galactosyl eosin, Mt6a/N-((9-beta-D -ribofuranosylpurin-6-yl)N-methyl-carbamoyl)threonine, Gm/2'-O-methylguanosine, Mv/uridine-5-oxyacetic acid methyl ester, I/inosine, o5u/uridine-5-oxy Acetic acid (v), I6a/N6-isopentenyladenosine, Osyw/Wy butoxocine, m1a/1-methyladenosine, P/pseudouridine, m1f/1-methylpseudouridine, Q/queosine, m1g/1-methylguanosine, s2c /2-thiocytidine, m1I/1-methylinosine, s2t/5-methyl-2-thiouridine, m22g/2,2-dimethylguanosine, s2u/2-thiouridine, m2a/2-methyladenosine, s4u/4-thiouridine, m2g/2-methylguanosine, T/5-methyluridine, m3c/3-methylcytidine, t6a/N-((9-beta-D-ribofuranosylpurin-6-yl)carbamoyl)threonine, m5c/5- Methylcytidine, Tm/2'-O-methyl-5-methyluridine, m6a/N6-methyladenosine, Um/2'-O-methyluridine, m7g/7-methylguanosine, Yw/Wy butocine, Mam5u/5- Examples include, but are not limited to, methylaminomethyluridine, or X/3-(3-amino-3-carboxypropyl)uridine, (acp3)u.

B. Nucleosides As used herein, "nucleoside" refers to an individual chemical unit that includes a nucleobase covalently attached to a nucleobase linker moiety. Non-limiting examples of "nucleobase linker moieties" are sugars containing 5-carbon atoms (i.e., "5-carbon sugars"), such as deoxyribose, ribose, arabinose, or derivatives or analogs of 5-carbon sugars. Examples include, but are not limited to. Non-limiting examples of derivatives or analogs of 5-carbon sugars include 2'-fluoro-2'-deoxyribose or carbocyclic sugars in which a carbon is replaced with an oxygen atom in the sugar ring. As used herein, "moiety" generally refers to a smaller chemical or molecular component of a larger chemical or molecular structure.

Different types of covalent attachment(s) of nucleobases to nucleobase linker moieties are known in the art. As a non-limiting example, a nucleoside containing a purine (i.e., A or G) or 7-deazapurine nucleobase is typically a purine at the 1' position of a 5-carbon sugar or a nucleoside at the 9 position of a 7-deazapurine. Contains covalent bonds. In another non-limiting example, a nucleoside that includes a pyrimidine nucleobase (i.e., C, T or U) typically includes a covalent bond of the 1 position of the pyrimidine to the 1' position of a 5-carbon sugar. (Kornberg and Baker, 1992)

C. Nucleotides As used herein, "nucleotide" refers to a nucleoside that further includes a "backbone linkage." Backbone linkages generally covalently link a nucleotide to another nucleotide-containing molecule or to another nucleotide to form a nucleic acid. A "backbone linkage" in naturally occurring nucleotides typically includes a phosphate moiety (eg, a phosphodiester backbone linkage) that is covalently linked to a 5-carbon sugar. Attachment of the backbone moiety typically occurs at either the 3' or 5' position of the 5-carbon sugar. However, other types of linkages are known in the art, particularly when the nucleotide contains derivatives or analogs of naturally occurring 5-carbon sugars or phosphate moieties.

D. Nucleic Acid Analogs Nucleic acids may contain, or consist entirely of, derivatives or analogs of nucleobases, nucleobase linker moieties, and/or backbone linkages that may be present in naturally occurring nucleic acids. As used herein, "derivative" refers to a chemically modified or altered form of a molecule of natural origin, whereas the term "mimetic" or "analog" refers to a molecule of natural origin or Refers to molecules with similar functions, which may or may not be structurally similar to the moieties. Nucleobases, nucleosides, and nucleotide analogs or derivatives are well known in the art.

Non-limiting examples of nucleosides, nucleotides, or nucleic acids containing 5-carbon sugars and/or backbone-linked derivatives or analogs include purines that form triple helices with dsDNA and/or prevent expression of dsDNA. U.S. Pat. No. 5,681,947, which describes oligonucleotides containing derivatives; U.S. Pat. No. 652,099 and No. 5,763,167; U.S. Pat. No. 5,614,617 describes oligonucleotide analogs with substituents on the pyrimidine ring with enhanced nuclease stability; Nos. 5,670,663 and 5,872,232, which describe oligonucleotide analogs with modified 5-carbon sugars (i.e., modified 2'-deoxyfuranosyl moieties) used No. 1, and No. 5,859,221; describes oligonucleotides containing at least one 5-carbon sugar moiety substituted at the 4' position with a substituent other than hydrogen that can be used in hybridization assays. US Pat. No. 5,446,137; US Pat. No. 5,886, which describes oligonucleotides having both deoxyribonucleotides with 3'-5' backbone linkages and ribonucleotides with 2'-5' backbone linkages. , 165; U.S. Patent No. 5,714,606 describes a modified backbone linkage in which the oxygen at the 3' position of the backbone linkage is replaced with a carbon, enhancing nuclease resistance of the nucleic acid; 1 No. 5,672,697, which describes oligonucleotides containing one or more 5' methylene phosphonate backbone linkages; Nos. 5,466,786 and 5,792,847, which describe the attachment of substituent moieties that may include a drug or label to the 2' carbon of; enhanced cellular uptake, resistance to nucleases, and targeting. U.S. Pat. No. 5,223,618; U.S. Patent No. 5,470,967 describing oligonucleotides containing at least one sulfamate or sulfamide backbone linkage useful as nucleic acid hybridization probes; improved nuclease resistance, cellular uptake , and U.S. Pat. No. 5,378,825, which describes oligonucleotides with 3- or 4-atom backbone binding moieties that replace phosphodiester backbone linkages for use in the regulation of RNA expression; No. 777,092, No. 5,623,070, No. 5,610,289, and No. 5,602,240; binds to the 2'-O position of oligonucleotides and improves their membrane permeability. and US Pat. No. 5,858,988 describing hydrophobic carrier factors that enhance stability; enhanced hybridization to DNA or RNA, enhanced stability against nucleases, and binding to anthraquinones at the 5' end. US Pat. No. 5,214,136 describes oligonucleotides that are 2'-deoxy-erythro-pentophala-containing for enhanced nuclease resistance, binding affinity, and ability to activate RNase H. US Patent No. 5,700,922, which describes a PNA-DNA-PNA chimera containing nosyl nucleotides; US Patent No. 5,708,154, which describes RNA that is attached to DNA and forms a DNA-RNA hybrid; US Pat. No. 5,908,845 describes polyether nucleic acids in which one or more nucleobases are attached to a chiral carbon atom in a polyether backbone; generally one or more nucleotides or nucleosides containing a nucleobase moiety , nucleobase linker moieties that are not 5-carbon sugars (e.g., aza nitrogen atoms, amide and/or ureido tethers), and/or backbone linkages that are not phosphate backbone linkages (e.g., aminoethylglycine, polyamide, polyethyl, polythioamide, U.S. Pat. No. 5,786,461; No. 5,786,461, No. 5,773,571, No. 5,766,855, No. 5,736,336, No. 5,719,262, No. 5,714 , 331, 5,539,082, and WO 92/20702; and US Pat. No. 5,855,911, which describes hydrophobic nuclease-resistant p-ethoxy backbone linkages.

Other modifications and uses of nucleic acid analogs are known in the art, and it is anticipated that these techniques and types of nucleic acid analogs can be used with the present invention.

E. Preparation of Nucleic Acids Nucleic acids can be made by any technique known to those skilled in the art, such as chemical synthesis, enzymatic production or biological production. Non-limiting examples of synthetic nucleic acids (e.g., synthetic oligonucleotides) include phosphotriesters, phosphites, such as those described in EP 266,032, incorporated herein by reference. or by in vitro chemical synthesis using phosphoramidite chemistry and solid phase technology, or by Froehler et al., each of which is incorporated herein by reference. (1986) and US Pat. No. 5,705,629. One or more oligonucleotides may be used in the methods of the invention. Various mechanisms of oligonucleotide synthesis are described, for example, in U.S. Pat. No. 4,959,463, No. 5,428,148, No. 5,554,744, No. 5,574,146, No. 5,602,244, and these Each is incorporated herein by reference.

F. Purification of Nucleic Acids Nucleic acids are purified by polyacrylamide gels, cesium chloride gradient centrifugation, or any other means known to those skilled in the art (see, e.g., Sambrook et al. (2001), incorporated herein by reference). be able to.

In certain embodiments, the invention relates to nucleic acids that are isolated nucleic acids. As used herein, the term "isolated nucleic acid" refers to a nucleic acid that has been isolated or otherwise isolated from the bulk of the whole genome and transcribed nucleic acids of one or more cells. refers to nucleic acid molecules (e.g., RNA or DNA molecules) that do not contain them. In certain embodiments, an "isolated nucleic acid" refers to an isolated nucleic acid that is free and isolated from the majority of cellular components or in vitro reaction components, such as macromolecules such as lipids or proteins, small biological molecules, etc. refers to a nucleic acid that has been or otherwise does not contain them.

G. Hybridization As used herein, "hybridization,""hybridize," or "capable of hybridizing" refers to double-stranded or triple-stranded molecules, or partially double-stranded or It is understood to mean the formation of a molecule with triple-stranded character. As used herein, the term "anneal" is synonymous with "hybridize."

As used herein, "stringent conditions" or "high stringency" refers to conditions between one or more nucleic acid strand(s) containing complementary sequence(s) or conditions that allow hybridization therein but exclude hybridization of random sequences. Stringent conditions tolerate little, if any, mismatch between the nucleic acid and the target strand. Such conditions are well known to those skilled in the art and are preferred for applications requiring high selectivity.

Stringent conditions can include low salt and/or high temperature conditions, such as those provided by about 0.02M to about 0.15M NaCl at a temperature of about 50°C to about 70°C. The desired stringency of temperature and ionic strength depends on the length of the particular nucleic acid(s), the length and nucleobase content of the target sequence(s), the charge composition of the nucleic acid(s), and the hybridization mixture. It is understood that the concentration of formamide, tetramethylammonium chloride or other solvent(s) in the solvent is determined in part.

These ranges, compositions and conditions for hybridization are mentioned merely as non-limiting examples, and the desired stringency for a particular hybridization reaction is often one or more positive or It is also understood that it is determined empirically by comparison with a negative control. Depending on the envisaged use, it is preferred to use varying hybridization conditions to achieve varying degrees of selectivity of the nucleic acid for the target sequence. In a non-limiting example, identification or isolation of relevant target nucleic acids that do not hybridize to nucleic acids under stringent conditions may be accomplished by hybridization at low temperatures and/or high ionic strength. Such conditions are referred to as "low stringency" or "low stringency conditions," and non-limiting examples of low stringency include temperatures ranging from about 0.15 M to about 20 °C to about 50 °C. Hybridization performed in about 0.9M NaCl is included. It is, of course, within the skill of those skilled in the art to further modify the low or high stringency conditions to suit a particular application.

IV. Method of Manufacturing Liposomal p-Ethoxy Antisense Formulation The liposomal p-ethoxy antisense formulation consists of two cGMP products, both of which have FDA-required certificates of analysis according to FDA-approved release criteria. Raw materials, solvents, and finished formulations are described herein. When manufactured, the formulation is an amber or white lyophilized crystal or powder containing the following materials: oligonucleotide (e.g., p-ethoxy antisense drug substance), neutral lipid (e.g., DOPC), and surfactant. agents (e.g. polysorbate 20). In preparation for administration to a patient, normal saline is added to the vial, at which point the p-ethoxy antisense is incorporated therein to form a liposome.

Certain physical properties of the final product (e.g., solubility and hydrophobicity, which in turn affect formulation solubility in saline, incorporation of oligos into liposomes, and liposome particle size) During the manufacture of the sense drug substance, a predetermined p-ethoxy and phosphodiester amidite raw material mix can be used and defined. An increase in the number of p-ethoxy molecules in the oligonucleotide backbone makes the molecule more hydrophobic (which results in larger liposome particles), less polar, and less soluble. As the oligonucleotide becomes less soluble due to more p-ethoxy backbone bonds, the reconstitution solution becomes whiter and eventually becomes too hydrophobic and particulate matter is formed.

The effect of surfactant (polysorbate 20) on liposome particle size was determined by titrating the amount of surfactant. In the absence of polysorbate 20, only 2.8% of the particles had a diameter of 300 nm or less. In the presence of 1x polysorbate 20 (approximately 5% of the total liposomal p-ethoxy antisense formulation), 12.5% of the particles had a diameter of 300 nm or less. By adding 3x to 10x polysorbate 20, approximately 20% of the particles had a diameter of 300 nm or less. Therefore, increasing surfactant from 1 to 3 times results in a decrease in particle size.

V. Test method for liposomal p-ethoxy antisense formulations Visual inspection of manufactured formulations: After manufacture, sample vials containing the formulation are selected and subjected to visual inspection. Absence of liquid is a prerequisite and the presence of amber crystals at the bottom of the vial is acceptable, extending acceptance to the best result, a white agglomerated powder or appearance. The white appearance suggests a good drying process and the high surface area to mass ratio greatly aids in reconstitution for use.

Visual inspection of reconstituted drug ready for patient IV: Add normal saline to the vial containing the prepared liposomal p-ethoxy antisense formulation and shake to ensure that drug crystals or powder are completely removed. Reconstitute into solution in dissolved state. Three main observations are made: 1) the crystals or powder are completely dissolved, 2) there are no white lumps of insoluble material, and 3) they are milky white or similar in appearance to skim milk. It's about appearance. The more bluish the appearance of the reconstituted solution, the better, as this is indicative of smaller liposome particle sizes that reflect light in the blue spectrum.

Mass spectrometry: Mass spec is used to display the profile of various masses in a sample. Once the p-ethoxy antisense material is produced, mass spectrometry is performed on the sample. The results show peaks of matter present on the grid with increasing mass to the right from the "x" axis and relative mass intensity on the "y" axis increasing upward. The profile from the sample is analyzed to determine the relative amount of p-ethoxy backbone in the p-ethoxy sample, and the profile of the peaks (starting from the right edge) is the full-length one with all backbones composed of p-ethoxy bonds. The next peak that represents the substance and moves to the left represents the full length with one backbone with a p-ethoxy deletion (thus the ethyl is removed, resulting in the normal phosphodiester backbone linkage). , and so on. A right-shifted mass spectrometry pattern represents a p-ethoxy sample with more p-ethoxy backbone and therefore more hydrophobic and less soluble properties; similarly, a left-shifted pattern represents represents a sample with opposite properties. Examination of the mass spectrometry chart of the sample can also be used to determine whether filtration during manufacturing has resulted in any adverse effects on the oligonucleotide composition present in the filtered formulation.

UV test: A UV test is used to determine the mass of oligonucleotides present in a sample. Oligonucleotides absorb light in the 260 nanometer range. As a result, UV testing of completed reconstituted formulations has come to be used as a method in determining the amount of oligonucleotide drug substance in a vial of formulation. In terms of manufacturing development and innovation, UV testing has improved due to problems encountered during filtration during manufacturing or defects in the p-ethoxy antisense drug substance, resulting in fewer oligonucleotides in solution and therefore lower UV readings. It was used to determine whether there is a certain solubility. The method will be validated and will likely become part of final product release testing.

Liposome particle size: Reconstitute vials of finished formulation and test for liposome particle size. The result is often an approximately normal distribution with a central point, boundary values and mean values, or a smaller secondary peak of the majority of particles and smaller liposome particles resulting from secondary particle formation effects. is approximately normal distribution. It is important that the liposome particles are not too large, as this can cause adverse effects in the patient (eg, causing blood flow problems in the smaller blood vessels of the lungs). Consequently, formulation release criteria include particle size testing showing that 90% of the liposomes are about 5 microns or smaller in size or about 3 microns or smaller in size. In addition, smaller liposomes are preferred because they appear to have better uptake into cells, and secondly, smaller liposomes are able to penetrate vascular pores, thereby , to allow liposomes to penetrate inside the tumor and increase the therapeutic efficacy of liposomal p-ethoxy antisense formulations.

VI. Methods of Treatment Certain embodiments of the invention provide oligonucleotide-lipid complexes (eg, oligonucleotides incorporated into uncharged liposomes) for treating diseases such as cancer, autoimmune diseases, or infectious diseases. In particular, the oligonucleotide has a sequence that allows base pairing with a human nucleotide sequence, thereby inhibiting the expression of the protein encoded by the human nucleotide sequence.

"Treatment" and "treating" refer to the administration or application of a therapeutic agent to a subject, or the treatment or treatment of a subject for the purpose of obtaining a therapeutic effect of a disease or health-related condition. For example, treatment can include administering a pharmaceutically effective amount of an oligonucleotide-lipid conjugate.

"Subject" and "patient" refer to either humans or non-humans, such as primates, mammals, and vertebrates. In certain embodiments, the subject is a human.

As used throughout this application, the terms "therapeutic effect" or "therapeutically effective" refer to anything that promotes or enhances the state of health of a subject with respect to medical treatment of the condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, treating cancer can include, for example, reducing the size of a tumor, reducing the invasiveness of a tumor, reducing the rate of cancer growth, or preventing metastasis. Treating cancer may also refer to prolonging the survival of a subject suffering from cancer. Treatment of autoimmune diseases includes, for example, reducing the expression of self-antigens against which there is an undesirable immune response, inducing tolerance to self-antigens against which there is an undesirable immune response, or inhibiting the immune response against self-antigens. may include. Treatment of an infectious disease can include, for example, eliminating the infectious agent, reducing the level of the infectious agent, or maintaining the level of the infectious agent at a certain level.

Tumors for which this treatment is useful include any malignant cell type, such as those found in solid tumors, hematological tumors, metastatic cancers, or non-metastatic cancers. Exemplary solid tumors include pancreas, colon, cecum, esophagus, gastrointestinal, gingiva, liver, skin, stomach, testis, tongue, uterus, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, Examples include, but are not limited to, tumors of organs selected from the group consisting of bone, lung, bladder, melanoma, prostate, and breast. Examples of hematological tumors include tumors such as bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myeloma, and the like. Additional examples of cancers that can be treated using the methods provided herein include carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell carcinoma, lung cancer (small cell lung cancer, non-small cell lung cancer, lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glial cancer Blastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine carcinoma, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, Thyroid cancer, various types of head and neck cancer, melanoma, superficial melanoma, lentiginous malignant melanoma, acral lentiginous melanoma, nodular melanoma, and B-cell lymphoma (low-grade/follicular non-Hodgkin Lymphoma (NHL); small lymphocytic (SL) NHL; intermediate/follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-incised cell NHL ; giant mass lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström hypergammaglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia , multiple myeloma, acute myeloid leukemia (AML), and chronic myeloblastic leukemia.

Cancers may be specifically, but not limited to, of the following histological types: malignant neoplasms; carcinomas; undifferentiated carcinomas; giant cell and spindle cell tumors; small cell carcinomas; papillary carcinomas; Epithelial carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilocytoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined carcinoma of hepatocellular carcinoma and cholangiocarcinoma; cord-shaped adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; familial polyposis adenocarcinoma of the colon; solid tumor; malignant carcinoid tumor; bronchioloalveolar epithelial adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; Acidic cell carcinoma; eosinophilic adenoma; basophilic adenoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical cell carcinoma; endometrium sexual tumor; skin adnexal tumor; apocrine adenocarcinoma; sebaceous gland carcinoma; ceruminous gland carcinoma; mucinous epidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous Adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory breast cancer; Paget's disease of the breast; acinic cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma ;Malignant ovarian stromal cell carcinoma;Malignant capsular cell carcinoma;Malignant granulosa cell carcinoma;Malignant cell carcinoma;Sertoli cell carcinoma;Malignant Leydig cell carcinoma;Malignant lipid cell carcinoma;Malignant paraganglioma;Malignant extramammary paraneural adenoma; pheochromocytoma; malignant glomus tumor (glomangiosarcoma); malignant melanoma; melanin-deficient melanoma; superficial spreading melanoma; malignant melanoma giant cell pigmented nevus; epithelioid cell melanoma; malignant blue mother Plaque; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonic rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal cell sarcoma; mixed Tumor; Mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal cell tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonic carcinoma ; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; paraosseous osteosarcoma; ; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; enamel epithelial sarcoma; Malignant glioma; ependymocytoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioblastoma undifferentiated neuroectodermal tumor; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant Granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's; lateral granuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specific non-Hodgkin's disease lymphoma; malignant histiocytic hyperplasia; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

Autoimmune diseases for which this treatment is useful include spondyloarthropathies, ankylosing spondylitis, psoriatic arthritis, reactive arthritis, enteroinflammatory arthritis, diabetes mellitus, childhood steatorrhea, autoimmune thyroid disease, and autoimmune diseases. Liver disease, Addison's disease, transplant rejection, graft-versus-host disease, host-versus-graft disease, ulcerative colitis, Crohn's disease, irritable bowel disease, inflammatory bowel disease, rheumatoid arthritis, juvenile rheumatoid arthritis, family including, but not limited to, Mediterranean fever, amyotrophic lateral sclerosis, Sjögren's syndrome, primary arthritis, viral arthritis, multiple sclerosis, or psoriasis. Diagnosis and treatment of these diseases are well described in the literature.

Infectious diseases for which this treatment is useful include, but are not limited to, bacterial, viral, fungal, and parasitic infections. Exemplary viral infections include hepatitis B virus, hepatitis C virus, human immunodeficiency virus 1, human immunodeficiency virus 2, human papillomavirus, herpes simplex virus 1, herpes simplex virus 2, herpes zoster, and varicella zoster. , Coxsackievirus A16, cytomegalovirus, Ebola virus, enterovirus, Epstein-Barr virus, hantavirus, Hendra virus, viral meningitis, respiratory rash virus, rotavirus, West Nile virus, adenovirus, influenza virus infection can be mentioned. Exemplary bacterial infections include Chlamydia trachomatis, Listeria monocytogenes, Helicobacter pylori, Escherichia coli, Borelia burgdorferi, Legionella pne umophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intraceliuia e, M. kansaii, M. .gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Streptococcus pyogenes (group A streptococcus), Streptococcus ag alactiae (group B streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic bacteria), Streptococcus pneumoniae, pathogenic Campylobacter sp. , Enterococcus sp. , Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, corynebacterium sp. , Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pastu Rella multocida, Bacteroides sp. , Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, Actinomy ces israelli, Shigella spp. (eg, S. flexneri, S. sonnei, S. dysenteriae), and Salmonella spp. Exemplary fungal infections include Candida albicans, Candida glabrata, Aspergillus fumigatus, Aspergillus terreus, Cryptococcus neoformans, Histoplasma caps ulatum, Coccidioides immitis, Blastomyces dermatitidis, and Chlamydia irachomatis infections.

The oligonucleotide-lipid conjugates herein can be used in a variety of therapeutics as antitumor, antiviral, antibacterial, antifungal, antiparasitic, or antiautoimmune agents. In certain embodiments, the invention provides methods of using oligonucleotide-lipid conjugates to target a population of diseased cells with a therapeutically effective amount of oligonucleotide-lipid conjugate and for a period of time sufficient to inhibit or reverse the disease. It is assumed that this includes contact.

In one embodiment, contacting in vivo comprises administering a therapeutically effective amount of a physiologically acceptable composition comprising an oligonucleotide-lipid conjugate of the invention to a patient by intravenous, intraperitoneal, subcutaneous, or intratumoral injection. This can be achieved by administering The oligonucleotide-lipid complex can be administered parenterally by injection or gradual infusion over time.

Therapeutic compositions containing oligonucleotide-lipid complexes are typically administered intravenously or subcutaneously, such as by unit dose injection. The term "unit dose" when used in reference to a therapeutic composition refers to physically discrete units suitable as a unit dose for a subject, each unit containing any required diluent, i.e., carrier or It contains a predetermined amount of active substance calculated to produce the desired therapeutic effect in association with the vehicle.

The compositions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The amount administered depends on the subject being treated, the ability of the subject's system to utilize the active ingredient, and the degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges for systemic administration are disclosed herein and depend on the route of administration. Suitable dosing schedules for initial and booster doses are also contemplated, with the initial dose typically followed by repeated administration by subsequent injections or other administrations at intervals of one hour or more. Exemplary multiple administrations are described herein and are particularly preferred for maintaining continuously high serum and tissue levels of the polypeptide. Alternatively, continuous intravenous infusion sufficient to maintain blood concentrations within the range specified for in vivo therapy is envisioned.

The oligonucleotides of the invention can be used systemically for disease treatment, such as to inhibit tumor cell growth or kill cancer cells in cancer patients suffering from locally advanced or metastatic cancer. It is envisioned that it may be administered locally or locally. They can be administered intravenously, intrathecally, subcutaneously and/or intraperitoneally. They can be administered alone or in combination with antiproliferative agents. In one embodiment, they are administered to reduce a patient's cancer burden prior to surgery or other treatment. Alternatively, they can be administered after surgery to ensure that any residual cancer (eg, cancer that could not be removed by surgery) does not survive.

A therapeutically effective amount of oligonucleotide is a predetermined amount calculated to achieve a desired effect, eg, to inhibit expression of a target protein. Thereby, the dosage range for administration of the oligonucleotides of the invention is one large enough to produce the desired effect. The dosage should not be so large as to cause adverse side effects such as hyperadhesive syndrome, pulmonary edema, congestive heart failure, neurological effects, etc. In general, the dosage will vary depending on the age, condition, sex, and extent of the disease of the patient and can be determined by one of ordinary skill in the art. Dosage may be adjusted by the individual physician if any complications occur.

The compositions of the invention are preferably administered to a patient parenterally, for example by intravenous, intraarterial, intramuscular, intralymphatic, intraperitoneal, subcutaneous, intrapleural, or intrathecal injection, or ex vivo. It can be used in The preferred dosage is 5-25 mg/kg. Administration is preferably repeated on a scheduled schedule until the cancer disappears or regresses, and can also be done in conjunction with other forms of therapy.

VII. Pharmaceutical Formulation Pharmaceutical compositions containing liposomes will typically include a sterile pharmaceutically acceptable carrier or diluent such as water or saline solution.

When clinical applications of uncharged lipid components containing oligonucleotides (e.g. in the form of liposomes) are made, it is generally beneficial to prepare the lipid complexes as pharmaceutical compositions suitable for the intended application. Dew. This will typically involve preparing a pharmaceutical composition essentially free of pyrogens and any other impurities that may be harmful to humans or animals. Appropriate buffers may be used that stabilize the complex and allow uptake by target cells.

The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse allergic or other unintended reactions when administered to animals, including humans, as appropriate. point to something Preparation of pharmaceutical compositions containing at least one uncharged lipid component with oligonucleotides or additional active ingredients, as exemplified by Remington: The Science and Practice of Pharmacy, 21st, 2005, incorporated herein by reference. will be known to those skilled in the art in view of this disclosure. Additionally, it will be appreciated that for administration to animals (eg, humans), the formulation must meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Products Standards.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives ( For example, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, Includes flavoring agents, dyes, such materials and combinations thereof. Pharmaceutically acceptable carriers are preferably formulated for administration to humans, and in certain embodiments, pharmaceutically acceptable carriers formulated for administration to non-human animals are used. Although this may be desirable, this may not be acceptable for human administration (eg, due to government regulations). Unless any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

The actual dosage of a composition of the invention administered to a patient or subject will depend on body weight, severity of the condition, type of disease being treated, previous or concomitant therapeutic interventions, the patient's underlying illness, and the duration of administration. may be determined by physical and physiological factors such as route. The health care professional responsible for administration will, in any event, determine the concentration of active ingredient(s) in the composition and the appropriate dose(s) for the individual subject.

In certain embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% active compound. In other embodiments, the active compound may constitute, for example, about 2% to about 75% of the unit weight, or about 25% to about 60%, and any derivable ranges therein. In other non-limiting examples, the dosage may also be about 1 microgram/kg/body weight, about 5 micrograms/kg/body weight, about 10 micrograms/kg/body weight, about 50 micrograms per administration. /kg/body weight, about 100 micrograms/kg/body weight, about 200 micrograms/kg/body weight, about 350 micrograms/kg/body weight, about 500 micrograms/kg/body weight, about 1 milligram/kg/body weight, about 5 milligrams/kg/body weight, about 10 milligrams/kg/body weight, about 50 milligrams/kg/body weight, about 100 milligrams/kg/body weight, about 200 milligrams/kg/body weight, about 350 milligrams/kg/body weight, about 500 milligrams /kg/body weight to about 1000 mg/kg/body weight or more, and any range derivable therein. Non-limiting examples of ranges that can be derived from the numbers recited herein include from about 5 μg/kg/body weight to about 100 mg/kg/body weight, from about 5 micrograms/kg/body weight to about 500 milligrams/kg/body weight. It can be administered within a range of body weight, etc.

The oligonucleotides of this embodiment are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70 per dose. , 80, 90, 100 μg or more of nucleic acid. Each dose may be 1, 10, 50, 100, 200, 500, 1000 μl or ml or more in volume.

Solutions of the therapeutic composition may be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under normal conditions of storage and use, these formulations contain preservatives to prevent the growth of microorganisms.

The therapeutic compositions of the invention are conveniently administered in the form of injectable compositions, either liquids or suspensions; solid forms suitable for solution in, or suspension in, prior to injection are also available. may be prepared. These formulations can also be emulsified. Typical compositions for such purposes include a pharmaceutically acceptable carrier. For example, the composition may contain up to 10 mg, 25 mg, 50 mg, or about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous non-toxic excipients including salts, preservatives, buffers, and the like.

Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate. Aqueous carriers include parenteral vehicles such as water, alcoholic/aqueous solutions, saline solutions, eg, sodium chloride, Ringer's dextrose, and the like. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobials, antioxidants, chelating agents, and inert gases. The pH and precise concentrations of the various components of the pharmaceutical composition are adjusted according to well-known parameters.

Therapeutic compositions of the invention may include typical pharmaceutical formulations. Administration of therapeutic compositions according to the invention will be via any conventional route so long as the target tissue is available via that route. Routes include oral, nasal, buccal, rectal, vaginal, or topical. Topical administration may be particularly advantageous in the treatment of skin cancer, to prevent chemotherapy-induced hair loss, or other skin hyperproliferative disorders. Alternatively, administration may be orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions will normally be administered as a pharmaceutically acceptable composition containing a physiologically acceptable carrier, buffer, or other excipient. Aerosol delivery can be used for the treatment of pulmonary conditions. The volume of the aerosol is about 0.01 ml to about 0.5 ml.

An effective amount of a therapeutic composition is determined based on the intended goals. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing the desired dosages described above in connection with its administration, i.e., the appropriate route and treatment regimen. contains a predetermined amount of the therapeutic composition calculated to produce a response of. The amount to be administered, both according to the number of treatments and the unit dose, depends on the desired protection and effect.

Precise amounts of therapeutic compositions also depend on the judgment of the health care professional and are peculiar to each individual. Factors that influence dosage include the patient's physical and clinical condition, the route of administration, the intended goal of treatment (e.g., symptom relief versus cure), and the efficacy, stability, and toxicity of the particular therapeutic agent. .

VIII. Combination Therapy In certain embodiments, the compositions and methods of the invention involve an inhibitory oligonucleotide, or an oligonucleotide capable of expressing an inhibitor of gene expression, in combination with a second or additional therapy. Methods and compositions involving combination treatments improve the therapeutic or protective effect and/or increase the therapeutic efficacy of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions may be provided in total amounts effective to achieve the desired effect, such as killing cancer cells and/or inhibiting cell hyperproliferation. This process can involve contacting the cell with both an inhibitor of gene expression and a second therapy. In this process, tissues, tumors, or cells are contacted with one or more compositions or pharmacological formulation(s) containing one or more drugs (i.e., inhibitors of gene expression or anticancer agents). or by contacting tissues, tumors, and/or cells with two or more separate compositions or formulations, wherein one composition contains 1) an inhibitory oligonucleotide, 2) an anticancer agent, or 3) Provide both inhibitory oligonucleotides and anticancer agents. It is also envisioned that such combination therapy may be used with chemotherapy, radiation therapy, surgery, or immunotherapy.

Inhibitory oligonucleotides may be administered before, during, after, or in various combinations with anti-cancer therapy. Administration may occur at intervals ranging from minutes to days to weeks after simultaneous administration. In embodiments where the inhibitory oligonucleotide is provided to the patient separately from the anti-cancer drug, a significant amount of time generally elapses between each delivery so that the two compounds can still beneficially exert a combined effect on the patient. This will ensure that it does not expire. In such cases, the inhibitory oligonucleotide therapy and the anticancer therapy are administered to the patient within about 12-24 or 72 hours of each other, more preferably within about 6-12 hours of each other. It is assumed that it can be provided to In some cases it may be desirable to significantly extend the time for treatment, here between several days (2, 3, 4, 5, 6 or 7 days) to several weeks. (1, 2, 3, 4, 5, 6, 7 or 8 weeks).

In certain embodiments, the series of treatments includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, Will last 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 days or longer . One drug is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, Administered on days 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and/or 90, or any combination thereof. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89th day, and/or 90th day, or It is envisaged that any combination of the following may be administered. During a day (24 hours), a patient may receive one or more doses of the drug(s). Furthermore, it is assumed that after a series of treatments, there will be a period during which anticancer drugs are not administered. This period may be 1, 2, 3, 4, 5, 6, 7 days and/or 1, 2, 3, 4, 5 weeks, depending on the patient's condition, e.g. their prognosis, physical fitness, health status, etc. , and/or may last for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer.

Various combinations may be used. For the examples below, inhibitory oligonucleotide therapy is "A" and anti-cancer drug therapy is "B".
A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A
A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B /B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of any compound or therapy of the invention to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the drug. Accordingly, in some embodiments there is a step of monitoring toxicity due to combination therapy. It is anticipated that treatment cycles will be repeated as necessary. It is also envisioned that various standard therapies, as well as surgical interventions, may be applied in combination with the described therapies.

In certain embodiments, standard therapy will include chemotherapy, radiation therapy, immunotherapy, surgical therapy, or gene therapy, and as described herein, therapy with inhibitors of gene expression, antibiotic therapy, etc. It is envisioned that it may be used in combination with cancer therapy or both therapy of inhibitors of gene expression and anti-cancer therapy.

A. Chemotherapy A wide variety of chemotherapeutic agents may be used according to the present embodiments. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to imply a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within the cell, eg, whether and at what stage they affect the cell cycle. Alternatively, drugs are characterized based on their ability to directly cross-link DNA, insert into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis. Good too.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, metledopa, and uredopa; altretamine, Ethylenimines and methylamelamines, including triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (particularly buratacin and buratacinone); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adzeresin, calzeresin, and vizeresin synthetic analogs); cryptophycin (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (synthetic analogs, KW-2189 and CB1-TM1); leutherobin; pancratistatin; sarcodictin; spongestatin; nitrogen mustards, such as chlorambucil, chlornafadine, chlorophosphamide, estramucin, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, mechlorethamine Faran, novenvitin, fenesterine, prednimustine, trophosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enedidine antibiotics (e.g. calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega I1); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamycin; Mycin, auslamycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino- (including doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, macelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogaramycin, olibomycin, pepromycin, potofilomycin , puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); such as denopterin, pteropterin, and trimetrexate. folic acid analogs; purine analogs such as fludarabine, 6-mercaptopurine, thiamipurine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; carsterone, androgens such as dromostanolone propionate, epithiostanol, mepithiostane, and testolactone; anti-adrenal agents such as mitotane and trilostane; folic acid replenishers such as fluoric acid; acegratone; aldophosphamide glycosides; aminolevulinic acid; maytansinoids such as eniluracil; amsacrine; bestrabcil; bisanthrene; edatraxate; defofamine; demecolcin; diazicon; erformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansine and ansamitocin; Mitoguazone; mitoxantrone; mopidamole; nitraerin; pentostatin; phenamet; pirarubicin; rosoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; , 2',2''-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, veraculin A, loridine A, and anguidine); urethanes; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobroman; Cyclophosphamide; taxoids such as paclitaxel and docetaxel; gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16) ; ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Included are retinoids; capecitabine; carboplatin; procarbazine; pricomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitor, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

B. Radiotherapy Other agents that cause DNA damage and have been widely used include gamma rays, commonly known as X-rays, and/or the directed delivery of radioactive isotopes to tumor cells. . Other forms of DNA damaging agents are also envisioned, such as microwaves, proton beam radiation (US Pat. Nos. 5,760,395 and 4,870,287), and UV radiation. All of these factors most likely affect widespread damage to DNA, to its precursors, to DNA replication and repair, and to chromosome assembly and maintenance. X-ray doses range from 50-200 roentgens per day over long periods of time (3-4 weeks) to single doses of 2000-6000 roentgens. The dose range for radioactive isotopes varies over a wide range and depends on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by neoplastic cells.

The terms "contacted" and "exposed" when applied to cells are used herein to mean that the therapeutic construct and chemotherapeutic or radiotherapeutic agent are delivered to the target cell and directly juxtaposed with the target cell. used to describe the process of emplacement. To achieve cell killing, for example, both agents are delivered to the cell in a total amount effective to kill the cell or prevent it from dividing.

C. Immunotherapy In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Trastuzumab (Herceptin™) is one such example. The immune effector may be, for example, an antibody specific for some marker on the surface of tumor cells. It may function as an effector of therapy, or it may recruit other cells to actually affect cell killing. , cholera toxin, pertussis toxin, etc.) and act simply as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts either directly or indirectly with the tumor cell target. A variety of effector cells include cytotoxic T cells and NK cells. A combination of therapeutic methods, i.e., direct cytotoxic activity and inhibition or reduction of ErbB2, may overactivate ErbB2. It will have a therapeutic effect in the treatment of developing cancer.

Other immunotherapies may also be used as part of combination therapy with the gene silencing therapies described above. In one aspect of immunotherapy, tumor cells should be able to be targeted, ie, carry some marker that is not present on most other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate-specific antigen, urinary tumor-associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor body, laminin receptor, erb B and p155. An alternative aspect of immunotherapy is to combine anticancer effects with immunostimulatory effects. Immune stimulating molecules including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand also exists. The use of immunostimulatory molecules in gene delivery, either in combination as proteins or in combination with tumor suppressors, has been shown to improve anti-tumor effects. Additionally, antibodies to any of these compounds can be used to target the anti-cancer agents discussed herein.

Examples of immunotherapies currently under investigation or in use include immunoadjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739). , 169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, such as interferon α, β, and γ; IL-1, GM-CSF and TNF (Bukowski et al., 1998; Davidson et al. al., 1998; Hellstrand et al., 1998) gene therapy, e.g. TNF, IL-1, IL-2, p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; US Pat. No. 5,830 , 880 and 5,846,945), and monoclonal antibodies such as anti-ganglioside GM2, anti-HER-2, anti-p185 (Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Pat. , 824, 311). It is envisioned that one or more anti-cancer therapies may be used in the gene silencing therapies described herein.

In active immunotherapy, antigenic peptides, polypeptides or proteins, or autologous or allogeneic tumor cell compositions or "vaccines" are administered, generally with a separate bacterial adjuvant (Ravindranath and Morton, 1991; Morton et al., Mitchell et al., 1990; Mitchell et al., 1993).

In adoptive immunotherapy, a patient's circulating or tumor-infiltrating lymphocytes are isolated in vitro, activated with lymphokines such as IL-2, or transduced with genes associated with tumor necrosis, and readministered. (Rosenberg et al., 1988; 1989).

D. Surgery Approximately 60% of people with cancer will undergo some type of surgery, including preventive, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that can be used in conjunction with other therapies such as the treatment of the present invention, chemotherapy, radiation therapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapy.

Curative surgery includes ablative procedures in which all or part of the cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery). It is further envisioned that the present invention may be used in conjunction with the removal of superficial cancers, precancerous lesions, or associated normal tissue mass.

A cavity may be formed within the body upon removal of all or part of a cancerous cell, tissue, or tumor. Treatment may be accomplished by irrigation, direct injection, or topical application of the area with additional anticancer therapy. Such treatments may be administered, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5 , may be repeated every 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also be of varying dosages.

E. Other Agents It is envisioned that other agents may be used in combination with certain aspects of this embodiment to improve the therapeutic effectiveness of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors as well as gap junctions, cytostatic and differentiating agents, inhibitors of cell adhesion, and agents that increase the susceptibility of hyperproliferating cells to apoptosis-inducing factors. , or other biological agents. The increase in cell-to-cell signaling by increasing the number of gap junctions will increase the anti-hyperproliferative effect on neighboring hyperproliferative cell populations. In other embodiments, cytostatic as well as differentiating agents can be used in combination with certain aspects of this embodiment to improve the anti-hyperproliferative efficacy of the treatment. It is envisioned that inhibitors of cell adhesion will improve the efficacy of this embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further envisioned that other agents that increase the susceptibility of hyperproliferative cells to apoptosis, such as antibody c225, can be used in combination with certain aspects of this embodiment to improve therapeutic efficacy.

IX. Kits and Diagnostic Methods Various aspects of the invention contemplate therapeutic kits containing therapeutic and/or other therapeutic agents and agents for delivery. In some embodiments, the invention contemplates kits for preparing and/or administering the therapies of the invention. The kit may contain reagents that can be used in administering the active agent or agent(s) of the invention. Reagents of the kit include at least one inhibitor of gene expression, one or more lipid components, one or more anti-cancer components of the combination therapy, and for preparing, formulating, and/or administering the components of the invention. or reagents for performing one or more steps of the methods of the invention.

In some embodiments, the kit also includes a suitable container means, such as an Eppendorf tube, assay plate, syringe, bottle, or tube, which is a container that is unlikely to react with the components of the kit. The container may be made from sterilizable materials such as plastic or glass.

The kit may further include instructions outlining the steps of the method procedure, which instructions may follow substantially the same procedures as described herein or known to those skilled in the art. Probably.

X. EXAMPLES The following examples are included to demonstrate preferred embodiments of the invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques found by the inventors to work well in the practice of the present invention and may therefore be considered to constitute a preferred mode of its practice. should be recognized by However, in view of this disclosure, those skilled in the art will recognize that many modifications can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention. Should.

Example 1 - Method of manufacturing a liposomal p-ethoxy antisense formulation A liposomal p-ethoxy antisense formulation is composed of two cGMP products. Both have FDA-required Certificates of Analysis with FDA-approved release standards. Raw materials, solvents, and finished formulations are described herein. When manufactured, the formulation is an amber or white lyophilized crystal or powder containing the following materials: oligonucleotide (e.g., p-ethoxy antisense drug substance), neutral lipid (e.g., DOPC), and surfactant. agents (e.g. polysorbate 20). In preparation for administration to a patient, normal saline is added to the vial, at which point the p-ethoxy antisense is incorporated therein to form a liposome.

ａ．このロットは、難溶性のために、具体的には、再構成溶液中のアンチセンス粒子のために廃棄された。
ｂ．このロットは、２０ｍＬのバイアル中、２ｍｇのアンチセンスと共に、より低いＤＭＳＯ及びｔＢＡ体積を有し、これは、リポソーム増大へのさらなる成分を付加した。
ｃ．このロットは、それが粒径リリース規格に達しなかったために、リリースされなかった。

ａ．このロットは、難溶性のために、具体的には、再構成溶液中のアンチセンス粒子のために廃棄された。
ｂ．このロットは、２０ｍＬのバイアル中、２ｍｇのアンチセンスと共に、より低いＤＭＳＯ及びｔＢＡ体積を有し、これは、リポソーム増大へのさらなる成分を付加した。
ｃ．このロットは、それが粒径リリース規格に達しなかったために、リリースされなかった。 p-ethoxy antisense drug substance. Certain physical properties of the final product (e.g., solubility and hydrophobicity, which in turn affect drug substance solubility in saline, incorporation of oligos into liposomes, and liposome particle size) During the manufacture of antisense drug substances, a predetermined p-ethoxy and phosphodiester amidite raw material mix can be used and defined. Although the loss of p-ethoxy backbone groups occurs randomly during the preparation of oligonucleotides, resulting in phosphodiester linkages in these linkages, this loss may alter the preferred ratio of p-ethoxy:phosphodiester backbone linkages in the oligonucleotide. It may not occur. In this case, the p-ethoxy and phosphodiester amidite raw material mix compensates for the expected deletion of the p-ethoxy backbone, thus producing an oligonucleotide with the desired proportions. An increase in the number of p-ethoxy molecules in the oligonucleotide backbone makes this molecule more hydrophobic (which results in larger liposome particles, Table 1), less polar, and less soluble (Table 1). 2). Test methods for charge-neutral hydrophobic p-ethoxy drug substances include mass spectrometry to determine the oligonucleotide length distribution and assays to determine the solubility of the drug substance; The practical purpose for this is visual inspection of the drug substance reconstituted in saline. As the oligonucleotide becomes less soluble due to more p-ethoxy backbone bonds, the reconstitution solution becomes whiter and eventually becomes too hydrophobic and particulate matter is formed.

a. This lot was discarded due to poor solubility, specifically antisense particles in the reconstitution solution.
b. This lot had a lower DMSO and tBA volume with 2 mg of antisense in a 20 mL vial, which added additional components to the liposome expansion.
c. This lot was not released because it did not meet particle size release specifications.

a. This lot was discarded due to poor solubility, specifically antisense particles in the reconstitution solution.
b. This lot had a lower DMSO and tBA volume with 2 mg of antisense in a 20 mL vial, which added additional components to the liposome expansion.
c. This lot was not released because it did not meet particle size release specifications.

Formulation, filtration, and lyophilization of liposomal p-ethoxy antisense formulations. One gram (1 g) of pE oligo is dissolved in DMSO at a ratio of 10 mg oligonucleotide per mL DMSO. Next, DOPC is added to the tert-butyl alcohol at a rate of 1 g DOPC per 1719 mL of tert-butyl alcohol. Oligo and DOPC are combined and mixed at a ratio of 1 g oligonucleotide per 2.67 g DOPC. 20 mL of a 0.835% (vol/vol) solution of polysorbate 20 is then added to this mixture resulting in a final concentration of 0.039 mg/mL. The solution is passed through a sterile filter before being dispensed into glass vials for lyophilization.

The effect of surfactant on liposome particle size was determined by titrating the amount of surfactant (Table 3). In the absence of polysorbate 20, only 2.8% of the particles had a diameter of 300 nm or less. In the presence of 1x polysorbate 20 (approximately 5% of the total liposomal p-ethoxy antisense formulation), 12.5% of the particles had a diameter of 300 nm or less. By adding 3x to 10x polysorbate 20, approximately 20% of the particles had a diameter of 300 nm or less. Therefore, an increase in surfactant from 1 to 3 times results in a decrease in particle size.

Preparation of liposomal p-ethoxy antisense formulations for administration. The lyophilized preparation was hydrated using normal saline (0.9%/10 mM NaCl) at a final oligo concentration of 10-5000 μM. Liposome-p-ethoxy oligo was mixed by hand shaking.

Example 2 - Testing method for liposomal p-ethoxy antisense formulations Visual inspection of manufactured formulations: After manufacture, sample vials containing the formulation are selected for visual inspection. Absence of liquid is a prerequisite and the presence of amber crystals at the bottom of the vial is acceptable, extending acceptance to the best result, a white agglomerated powder or appearance. The white appearance suggests a good drying process and the high surface area to mass ratio greatly aids in reconstitution for use.

Visual inspection of reconstituted drug ready for patient IV: Add normal saline to the vial containing the prepared liposomal p-ethoxy antisense formulation and shake to ensure that drug crystals or powder are completely removed. Reconstitute into solution in dissolved state. Three main observations are made: 1) the crystals or powder are completely dissolved, 2) there are no white lumps of insoluble material, and 3) they are milky white or similar in appearance to skim milk. It's about appearance. The more bluish the appearance of the reconstituted solution, the better, as this is indicative of smaller liposome particle sizes that reflect light in the blue spectrum.

Mass spectrometry: Mass spec is used to display the profile of various masses in a sample. Once the p-ethoxy antisense material is produced, mass spectrometry is performed on the sample. The results show peaks of material present on the grid with increasing mass to the right from the "x" axis and relative mass intensity on the "y" axis increasing upward. The profile from the sample is analyzed to determine the relative amount of p-ethoxy backbone in the p-ethoxy sample, and the profile of the peaks (starting from the right edge) is the full-length one with all backbones composed of p-ethoxy bonds. The next peak that represents the substance and moves to the left represents the full length with one backbone with the p-ethoxy deletion (thus the ethyl is removed, resulting in the normal phosphodiester backbone linkage). , and so on. A right-shifted mass spectrometry pattern represents a p-ethoxy sample with more p-ethoxy backbone and therefore more hydrophobic and less soluble properties; similarly, a left-shifted pattern represents a represents a sample with opposite properties. Examination of the mass spectrometry chart of the sample can also be used to determine whether filtration during manufacturing has resulted in any adverse effects on the oligonucleotide composition present in the filtered formulation.

UV test: A UV test is used to determine the mass of oligonucleotides present in a sample. Oligonucleotides absorb light in the 260 nanometer range. As a result, UV testing of completed reconstituted formulations has come to be used as a method in determining the amount of oligonucleotide drug substance in a vial of formulation. In terms of manufacturing development and innovation, UV testing has improved due to problems encountered during filtration during manufacturing or defects in the p-ethoxy antisense drug substance, resulting in fewer oligonucleotides in solution and therefore lower UV readings. It was used to determine whether there is a certain solubility. The method will be validated and will likely become part of final product release testing.

Liposome particle size: Reconstitute vials of finished formulation and test for liposome particle size. The result is often an approximately normal distribution with a center point, boundary values and mean values, or a smaller secondary peak of the majority of particles and smaller liposome particles resulting from secondary particle formation effects. is approximately normal distribution. It is important that the liposome particles are not too large, as this can cause adverse effects in the patient (eg, causing blood flow problems in the smaller blood vessels of the lungs). As a result, formulation release criteria include particle size testing showing that 90% of the liposomes are about 5 microns or smaller in size. In addition, smaller liposomes are preferred because they appear to have better uptake into cells, and secondly, smaller liposomes are able to penetrate vascular pores, thereby , to allow liposomes to penetrate inside the tumor and increase the therapeutic efficacy of liposomal p-ethoxy antisense formulations.

All of the methods disclosed and claimed herein can be made and practiced without undue experimentation in light of this disclosure. Although the compositions and methods of the invention have been described with respect to preferred embodiments, modifications can be made to the methods and methods described herein without departing from the concept, spirit and scope of the invention. It will be clear to those skilled in the art that it may be applied in a step or in a series of steps. More specifically, it will be clear that certain agents that are both chemically and physiologically related may be substituted for the agents described in this invention, so long as the same or similar results are obtained. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
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Claims (29)

A composition comprising a population of oligonucleotides, a phospholipid, and a surfactant, wherein the oligonucleotides of the population are composed of nucleoside molecules linked together through phosphate backbone bonds, 60% to 75% of the phosphate backbone bonds are p-ethoxy backbone bonds, and 25% to 40% of the phosphate backbone bonds in each oligonucleotide are phosphodiester backbone bonds,
the phospholipid is phosphatidylcholine, the surfactant is polysorbate 20, the oligonucleotide, phospholipid, and surfactant form liposomes, and at least 90% of the liposomes have a diameter of 5. The composition is less than a micron . 2. The method according to claim 1, wherein 65% to 71 % of the phosphoric acid skeletal bonds are p-ethoxy skeletal bonds, and 29% to 35% of the phosphoric acid skeletal bonds are phosphodiester skeletal bonds. Compositions as described. 2. According to claim 1, 60% to 70% of the phosphate backbone bonds are p-ethoxy backbone bonds, and 30% to 40% of the phosphate backbone bonds are phosphodiester backbone bonds. Composition of. The composition of claim 1, wherein the oligonucleotides of the population have a size ranging from 7 to 30 nucleotides. 2. The composition of claim 1, wherein the population of oligonucleotides comprises a single species of oligonucleotide. 2. The composition of claim 1, wherein the population of oligonucleotides comprises at least two species of oligonucleotides. 2. The composition of claim 1, wherein the population of oligonucleotides comprises antisense oligonucleotides, short interfering RNAs, microRNAs, or piwiRNAs. 2. The composition of claim 1, wherein the oligonucleotides of the population inhibit expression of at least one oncogenic protein, infectious agent protein, or autoantigen. 2. The composition of claim 1, wherein the oligonucleotides of the population hybridize with at least one oncogenic oligonucleotide, infectious agent oligonucleotide, or autoantigen oligonucleotide. 2. The composition of claim 1, wherein the phospholipid is uncharged or has a neutral charge at physiological pH. The composition according to claim 1, wherein the phospholipid is a neutral phospholipid. 12. The composition of claim 11, wherein the neutral phospholipid is dioleoylphosphatidylcholine. 2. The composition of claim 1, wherein the phospholipid is essentially free of cholesterol. 2. The composition of claim 1, wherein the phospholipid and oligonucleotide are present in a molar ratio of about 5:1 to about 100:1. 2. The composition of claim 1, wherein the composition is further defined as a population of liposomes. 16. The composition of claim 15 , wherein the population of oligonucleotides is incorporated into the population of liposomes. 2. The composition of claim 1, wherein the phosphodiester backbone linkages within each of the oligonucleotides of the population are randomly arranged throughout each oligonucleotide. 2. The composition of claim 1, wherein the composition is lyophilized. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier. 20. A pharmaceutical composition according to claim 19 , characterized in that the pharmaceutical composition is contacted with the cells in order to deliver the oligonucleotide to the cells. 21. The pharmaceutical composition according to claim 20 , wherein the pharmaceutical composition is a composition for treating hyperplasia, cancer, autoimmune disease, or infectious disease. 20. A pharmaceutical composition according to claim 19 for treating a subject having cancer, an autoimmune disease, or an infectious disease, characterized in that said pharmaceutical composition is administered to said subject. Pharmaceutical composition. 23. The pharmaceutical composition according to claim 22 , wherein the subject is a human. The cancer is bladder, blood, pancreatic, bone, bone marrow, brain, breast, colon, esophagus, stomach, head and neck, kidney, liver, lung, prostate, skin, testicular, tongue, ovarian, or uterine cancer. Pharmaceutical composition according to item 22 . 23. The pharmaceutical composition of claim 22 , wherein the autoimmune disease is lupus erythematosus, Sjögren's disease, Crohn's disease, diabetes mellitus, multiple sclerosis, or rheumatoid arthritis. 23. The pharmaceutical composition according to claim 22 , wherein the infectious disease is a bacterial infection, a fungal infection, a viral infection, or a parasitic infection. Pharmaceutical composition according to claim 22 , characterized in that the pharmaceutical composition is administered subcutaneously, intravenously or intraperitoneally. 23. Pharmaceutical composition according to claim 22 , characterized in that the pharmaceutical composition is administered in conjunction with at least a second anti-cancer therapy to the subject. The second anticancer therapy may include surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, antiviral therapy, immunosuppressive therapy, antibacterial therapy, antiparasitic therapy, antifungal therapy, or cytokine therapy. 29. A pharmaceutical composition according to claim 28 , which is a therapy.